This is the National Stage Filing of PCT/DE98/03437 filed Nov. 20, 1998 and published Jun. 3, 1999.
1. Field of the Invention
The present invention relates generally to a structure, on the surface of the support material of which structure molecular layers are immobilized so as to be electrically addressable, a method for the electrically addressable immobilization of molecules, a device for carrying out this method, and the use of this structure as a chemo- and/or biosensor, in particular as a multisensor system for chemical, biological, and physical assays, and for applications in the combinatorial synthesis on the boundary surface.
2. Prior Art
Both chemosensors and biosensors, i.e., comparatively small measuring arrangements, are increasingly needed to make it possible to carry out chemical or biochemical analyses rapidly and at the site of the occurrence. Compared to the immunological assay systems, they have the special advantage that they are able to quantify, preferably continuously, for example, concentrations of (bio)chemical substances within a short period of time and without a time-consuming preparation of the samples. A biosensor should therefore be small, which offers the additional advantage that it can be used in the vicinity of the site of the analysis; for example, using enzymatic biosensors, diabetics are able to determine their blood sugar level within a few minutes.
Thus, chemo- and biosensory analysis includes both detection and quantification. The process of molecular detection, for example, plays an important role in biology. For example, a cell should be able to detect a potassium ion and should not mistake it for a sodium ion which is very similar [to the potassium ion], the enzyme glucose dehydrogenase should only degrade glucose but not fructose, and the immune system should be able to recognize foreign organisms and substances that have invaded the system before it attacks them. This means that molecules that recognize the targeted substances are needed to make it possible to develop chemo- and biosensors. At the same time, the many other substances that exist side by side with the targeted ones must be ignored. Thus, a biosensor must be able to meet the following requirement, i.e., that the process of recognizing/coupling can be detected and xe2x80x9ctranslatedxe2x80x9d into a measurable signal. During this xe2x80x9ctranslation,xe2x80x9d however, serious problems arise so that there is a need for a system that makes detection easy. In systems that are used in the form of monosystems, it is not necessary to immobilize the molecules since only one single species has to be recognized and identified. If, on the other hand, multiple systems are to be used, such as multisensor systems, with which different molecules are to be detected at the same time, it is necessary to ensure an addressable immobilization of the molecular layers on the surface of the support material, thereby making it possible to detect the recognition of a signal. This type of system is not known in prior art.
In prior art, several methods for an addressable immobilization are available, with which it is possible to specifically couple several different types of molecules to a surface of the support material. Yershov et al. (Yershov, G., Barksy, V., Belgovskiy, A., Kirillov, E., Kreindlin, E., Ivanov, I., Parinov, S., Guschin, D., Drobishev, A., Dubiley, S., Mirzabekov, A., Proc. Natl. Acad. Sci. 43 (1996), pp. 4913-4916) as well as Blanchard et al. (Blanchard, A. P., Kaiser, R. J., Hood, L. E., Biosens. and Bioelectron. 11 (1996), pp. 687-690) describe the use of this technique, for example, to produce DNA arrays. In addition, these techniques are used for the micromechanical addressability and for the optical addressability. U.S. Pat. No. 5,412,087 describes the optical immobilization by coupling functional groups to photosensitive protecting groups. The system is activated by cleaving off the protecting groups by means of photolytic degradation. Chrisey et al. (Chrisey, L. A., O""Ferall, E., Spargo, B. J., Dulcey, C. S., Calvert, J. M., Nucl. Acids Res. 24 (1996), pp. 3040-3047) describe a different approach according to which the adsorption is carried out using the photoresist technique.
The known immobilization methods mentioned above, however, have many drawbacks which limit their applicability. Thus, for example, the resolution of the micromechanical immobilization is limited by the size of the individual spray particles. In the above-described method used by Yershov et al. (1996) and Blanchard et al. (1996), at best an optical resolution of approximately 100 xcexcm is possible. During the optical immobilization according to U.S. Pat. No. 5,412,087, it is always necessary to work with protecting groups which require that specific conditions (such as solvents, darkening the work area because the protecting groups are sensitive to light, etc.) be maintained and which must be removed at a certain point in time. The above-mentioned photoresist technique by Chrisey et al. (1996) is very time- and cost-consuming since a large number of photomasks must be used.
Thus, the problem to be solved by the present invention is to make available a structure which is able to both bind and detect molecules and thus to make them qualitatively and quantitatively determinable. An additional problem to be solved by the present invention is to make available a method that makes it possible for molecules to be electrically addressably immobilized or desorbed on surfaces and thus to avoid the drawbacks mentioned. In addition, yet another problem to be solved by the present invention is to design a device for carrying out the method described by this invention and thus for manufacturing the structure described by this invention.
This problem is solved by making available a structure with electrically addressable immobilized molecules, which structure comprises a support material, one and/or a plurality (1 to n) of electrically conductive support surface(s) which is/are located on this support material, and one and/or a plurality of immobilized identical and/or different receptor(s).
Although it is known that it is possible to influence the chemical adsorption of molecules on an electrode by changing the applied chemical potential and that this dependence on the potential also applies to the chemical adsorption of thiol compounds on electrodes, the work that led to the present invention focused on the conditions under which, for example, the coupling between gold and sulfur is stable. Surprisingly, however, it was discovered that a chemically adsorbed molecular layer on an electrode is stable only within a specific pH-dependent range of potential. These results go far beyond the findings that have been described in the pertinent literature by Imabyashi et al. (Imabyashi, S., Iida, M., Hobara, D., Feng, Z. Q., Niki, K., Kakiushi, T., J. Electroanal. Chem. 428 (1997), pp. 33-38).
The problem of making available a method for the manufacture of a structure of the type mentioned above is solved by addressably immobilizing molecules on surfaces in a simply manner. This method comprises the following steps:
(a) a support material with n electrodes, where n stands for an integral number, is introduced into a flow-type cell which contains the electrolyte solution and the molecules to be immobilized (receptors),
(b) either an adsorption potential or a desorption potential is applied to each separate electrode, as a result of which the uncoated electrode regions are kept inert by the desorption potential, and the adsorption potential that has already been applied to the coated electrode regions prevents the desorption of molecules as well as the adsorption of molecules that have a different structure, which ensures that the electrode region is occupied by the first type of molecules and the molecules are immobilized and securely coupled to the surface of the support material, and
(c) the adsorption of molecules on the electrode(s) is measured as a change in the signal.
The process is concluded when a constant signal value is reached, e.g., a capacitance, impedance, or resonant frequency value.
The method described by this invention has the advantage that it makes it possible to control the desorption or adsorption of molecules by means of changing the potential within as well as outside the potential stability range. Based on this principle, it is possible to form a specific lateral structure of the chemically adsorbed layer.
This method can be carried out using the device according to this invention which comprises an exchangeable device (1) for sample holders, a pump (2), a flow-type cell (3) into which the molecule or molecules desired or the receptor or receptors is/are transported by means of the pump (2), a support material (5) to which n electrodes are attached, with the support material (5) with n electrodes being located in the flow-type cell (3), a multiplexer (6), an address bus (7) which controls the multiplexer (6), as a result of which either an adsorption potential (8) or a desorption potential (9) is separately applied to the electrode(s) with respect to the reference electrode (10), and a collecting vessel (4) into which the excess molecules are pumped by means of the pump (2) once the molecules have been immobilized.
The structure according to this invention is used as a chemo- and/or biosensor, in particular as a multisensor system.
As such, the structure is preferably used directly in the sample medium, thus making an easy assay and/or detection possible by simply measuring the change in the signal value, in particular the capacitance, impedance, or resonant frequency value. In this manner, a system is made available by means of which the problems and disadvantages of the prior art systems mentioned above are avoided.
The structures according to this invention with the electrically addressable immobilized molecules can also be used for the combinatorial synthesis by specifically releasing moleculesxe2x80x94which are coupled to the surface of the electrode and which, depending on the electrode, can be identical to or different from each otherxe2x80x94by applying the desorption potential and thus by letting these molecules function as reaction participants during the synthesis step carried out or, if the synthesis is carried out on the surface, making it possible for the product to be cleaved off directly.
For the electrically addressable immobilization, any number of electrodes of random size and shape, which have been applied to a support material made of a dielectric material, can be used, and optionally, electrical leads for sensor spots can be incorporated into this dielectric material. The immobilization is carried out as shown in the diagrams of FIGS. 1 and 2 which will be described in greater detail below. During immobilization, different receptors (molecules A, B, C, etc.) which contain one or more thiol groups are specifically coupled to different electrodes (1, 2, 3, etc): receptor A to electrode E1, receptor B to electrode E2, etc. To obtain these couplings, the following steps are required: In a liquid electrolyte solution, a suitable electrode potential (adsorption potential) is applied to electrode E1 with respect to a reference electrode which supports the coupling to thiol groups. At the same time, an electrode potential (desorption potential) is applied to the other electrodes (2, 3, etc.) with respect to the same reference electrode which is able to suppress the coupling of the thiol groups to these electrodes. As a result, the added receptor molecule A is attached to electrode E1 only. Subsequently, the liquid electrolyte with the receptor molecules A is replaced with a liquid electrolyte solution with receptor molecules B, and these molecules are addressed to the next electrode site 2. By applying the desorption potential, the uncoated electrode sites are kept inert. The adsorption potential that is applied to the already coated electrode sites prevents the desorption of the molecules as well as the adsorption of differently structured molecules, since the electrode site is completely occupied by the first type of molecules. This process is repeated until the structure of the sensor has been completely built up. Between the immobilized molecule and the electrode, a permanent coupling is established. Thus, it is possible to produce a biosensor or a multisensor system (xe2x80x9carrayxe2x80x9d) with any molecular structure desired.
An additional benefit of the method described by the present invention pertains to the fact that, following the electrically addressable immobilization, it is possible for one or a plurality of molecular layer(s), e.g., antibodies or DNA strands, to be adsorbed and/or immobilized by way of functional groups to a random number (0 to n) of electrodes on which the receptors are located. For this purpose, the adsorption potential is maintained for all coated electrodes, and the liquid electrolyte solution of the molecules that are to be attached, in combination with a coupling reagent, are pumped simultaneously into the flow-type cell. As an alternative, it is also possible to first add the coupling reagent and then to add the molecules that are to be attached only once the activation of the base layer(s) has been concluded. If the molecules are able to attach themselves to the addressed layer on their own, there is no need for the coupling reagent, such as is the case with the avidin-biotin system, Ni-His tag, or hydrophobic-hydrophobic interactions of liposomes with unfunctionalized alkanethiol chains. After the molecules have been chemically or physically attached to the base layer(s), a liquid electrolyte solution is used to rinse and thus to remove unused or deactivated coupling reagent and/or uncoupled molecules from the cell. Depending on what field of application the detector system is to be used in, this process can be repeated several times until the desired structure of the sensor has been obtained.
Similarly, it is possible to coat two or more electrodes of the multisensor system (xe2x80x9carrayxe2x80x9d) by means of the adsorption potential and optionally modify them by means of a subsequent physical or chemical adsorption or coupling to one or several identical or different layers of molecules. Thus, if the desorption potential is applied to specific electrodes, these electrodes can be coated as described above with a different coating after the molecules have been desorbed and the desorbed molecules have been rinsed off. This process can be repeated until the multisensor structure desired has been obtained.
Suitable coupling reagents include, in particular, carbodiimides and derivatives thereof and N-succinimides and derivatives thereof.
Using the systems described above, it is possible to perform a screening procedure while carrying out different tests at the same time, thus making it possible to avoid having to carry out the separate and time-consuming screening procedures thus far required.
Suitable support materials include any solid dielectric substrate, in particular silicon, glass, any nonconductive synthetic material, such as Teflon, PVC, or PE, and any conductive or semiconductive substrate which is insulated from the electrode(s) by means of a dielectric layer.
The electrodes used are thin conductive materials which are intimately attached to the support material. In particular, suitable materials include Au, Pd, Pt, Ag, alloys, such as Au/Pd, Au/Ag, Ag/Pd, GaAs, and similar materials, doped semiconductors, and any other conductive or semiconductive inorganic or organic material, such as TCNQ, or TTF.
The reference electrodes used are electrodes generally used in electrochemistry, such as Ag/AgCl, etc., with and without a salt bridge.
The receptors, which are coupled to the electrodes by means of the process for the electrically addressable immobilization according to this invention, are molecules, the coupling of which to the electrodes can be controlled by the electrode potential. In most cases, these molecules contain a minimum of one thiol group or are coupled to a minimum of one thiol group or contain a sulfide or disulfide group. These molecules are selected from the group that comprises
HSxe2x80x94(CN2)nxe2x80x94X, where n stands for a number from 2 to 24 and X stands for H, OH, SH, CH3, COOH, NH2, and any other molecular fragment, xe2x80x94Xxe2x80x94(CH2)nxe2x80x94Sxe2x80x94Sxe2x80x94(CH2)mxe2x80x94Y or Xxe2x80x94(CH2)nxe2x80x94Sxe2x80x94(CH2)mxe2x80x94Y, where m and n stand for a number from 2 to 24 and X and Y stand for H, OH, SH, CH3, COOH, NH2 and any other molecular fragment, especially xcfx89-mercapto acids, n-alkanethiols, such as octanethiol, as well as toxins, hormones, hormone receptors, peptides, proteins, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, DNA, RNA, viruses, bacteriophages, prions, oligosaccharides, natural and artificial receptors, redox-active substances, dyes, acids, bases, epitopes, antigens or antibodies which contain a minimum of one thiol group or which are coupled to a minimum of one thiol-containing compound. Similarly, these receptors may contain sulfide or disulfide groups. The thiol groups may be present in such molecules from the very beginning or they may have been introduced later by chemical modification. In addition, in all of the compounds mentioned above, an Se atom may be substituted for the S atom of the thiol group.
The molecules that may be additionally attached to the receptors are selected from the group that comprises toxins, hormones, hormone receptors, peptides, proteins, enzymes, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, DNA, RNA, viruses, bacteriophages, prions, oligosaccharides, natural and artificial receptors, redox-active substances, dyes, acids, bases, epitopes, antigens or antibodies which contain a minimum of one functional group or which are able to enter into interactions with the adsorbed receptor, which makes it possible for them to be chemically and/or physically adsorbed and/or immobilized on the electrode(s).
As defined here, the adsorption potential is an electrical potential, the use of which supports and/or maintains the coupling of the molecule to the electrode. When thiols are adsorbed on electrodes, the adsorption potential at a pH values from 4.0 to 8.0 is in a range from 0 mV to +600 mV with respect to the Ag/AgCl electrode in 100 mM KCl, preferably the adsorption potential is approximately +300 mV. If the pH value is alkaline, the range of the adsorption potential shifts.
As defined here, the desorption potential is a potential outside the stability range defined above. The desorption potential must be sufficiently high to prevent the adsorption of molecules that are used during the immobilization process. Within a range of pH values from 4.0 to 8.0, the desorption potential is in a range beginning at xe2x88x92300 mV or lower with respect to the Ag/AgCl electrode in 100 mM KCl, preferably in a range from xe2x88x92600 mV to xe2x88x921800 mV and especially in a range from xe2x88x92800 mV to xe2x88x921400 mV.
Electrolyte solutions to be used include all aqueous solutions and organic electrolytes and mixtures thereof as well as a mixture of aqueous electrolytes and organic solvents or organic electrolytes and organic solvents.